Method of increasing plant transformation frequency using modified strains of Agrobacteria

Patent metadata

Field	Value
Publication number	US-9617551-B2
Application number	US-201113812469-A
Country	US
Kind code	B2
Filing date	Jul 29, 2011
Priority date	Jul 29, 2010
Publication date	Apr 11, 2017
Grant date	Apr 11, 2017

Abstract

Agrobacterium strains that harbor transformation-enhancing genes on a plasmid capable of replication independently of the Agrobacterium chromosome, the Ti plasmid, and plant transformation binary vectors, and uses for these Agrobacterium strains are provided. Additionally, Agrobacterium strains that are deficient in DNA recombination functions that result in instability or rearrangement of plant transformation binary vectors, and that harbor transformation-enhancing genes on a plasmid capable of replication independently of the Agrobacterium chromosome, the Ti plasmid, and plant transformation binary vectors, and uses for these strains, are also provided. Further included are Agrobacterium strains that harbor transformation-enhancing genes integrated into the Agrobacterium chromosome at a locus that does not interfere with or otherwise compromise the normal growth and plant transformation ability of the Agrobacterium cells, and uses for these Agrobacterium strains. Plants made using these Agrobacterium strains are also described.

First claim

What is claimed is:

1. A method for transforming a plant, comprising contacting a cell of the plant with an Agrobacterium strain that is deficient in Rec A function, wherein the Agrobacterium strain comprises:
at least one pTi helper plasmid comprising a 14.8 KpnI VirBCDG fragment of pSB1; and
a different pTi plasmid comprising at least one disarmed T-DNA region that includes (i) at least a right T-DNA border and (ii) exogenous DNA adjacent to the border, wherein the pTi helper plasmid and the pTi plasmid comprising at least one disarmed T-DNA region have differing origins of replication relative to each other, thus eliminating the need to form a co-integrant hybrid vector (HV) containing both the 14.8 KpnI fragment and the exogenous DNA.

2. The method for transforming a plant according to claim 1 , wherein the different pTi piasmid has a replication origin of an IncP incompatibility group.

3. The method for transforming a plant according to claim 2 , wherein the T-DNA region contains three or more gene sequences.

4. The method for transforming a plant according to claim 2 , wherein the T-DNA region contains equal to or greater than 25,000 nucleotide base pairs.

5. The method for transforming a plant according to claim 2 , wherein the T-DNA region is inserted into a single location in the plant cell when the plant is transformed.

6. The method for transforming a plant according to claim 2 , wherein the T-DNA region encodes one or more of an insecticidal protein, a herbicidal protein, or a mixture of insecticidal proteins and herbicide tolerance proteins.

7. The method for transforming a plant according to claim 2 , wherein the T-DNA region encodes a Cry1Ca insecticidal protein, a Cry1F insecticidal protein, and a Cry1Ab1 insecticidal protein.

8. The method for transforming a plant according to claim 2 , wherein the T-DNA region encodes a Cry1Ca insecticidal protein, a Cry1F insecticidal protein, a Cry1Ab1 insecticidal protein, and an AAD-1 herbicide tolerance protein.

9. The method according to claim 1 , wherein the plant is a monocot.

10. The method according to claim 1 , wherein the 14.8 KpnI VirBCDG fragment is cloned into the Kpn I site of a pDAB9291 plasmid.

11. The method according to claim 1 , wherein the pTi helper plasmid is plasmid pMP90.

12. The method according to claim 1 , wherein the pTi helper plasmid is plasmid pTiC58Δ.

13. The method according to claim 10 , further comprising transforming the Agrobacterium strain using plasmid pDAB9292 DNA.

14. The method according to claim 1 , further comprising a step of selecting a transformed cell or a transformed tissue, after subjecting said cultured tissue to transformation.

Assignees

• Merlo Donald J
• Russell Sean M
• Retallack Diane M
• Woosley Aaron T
• Meade Thomas
• Narva Kenneth
• Dow Agrosciences Llc

Inventors

• Merlo Donald J
• Russell Sean M
• Retallack Diane M
• Woosley Aaron T
• Meade Thomas
• Narva Kenneth

Classifications

• C12N15/8286

  for insect resistance · CPC title

• C12N15/8274

  for herbicide resistance · CPC title

• C12N15/8205 Primary

  Agrobacterium mediated transformation · CPC title

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• C12N15/743

  for Agrobacterium; Rhizobium; Bradyrhizobium · CPC title